Over the last decade, chemokines have emerged as key mediators of inflammation as a result of their numerous proinflammatory activities which affect virtually every leukocyte type. More recently, chemokines have been recognized as a critical component of basal leukocyte trafficking essential for normal immune surveillance and response, as well as for several other functions in hematopoiesis, angiogenesis, control of viral infection, and T cell differentiation (Baggiolini et al., Ann. Rev. Immunol 15:675 (1997); Zou et al., Nature 393:595 (1998); Tachibana et al., Nature 393:591 (1998)). This diverse array of biological activities, including mediation of a range of pro-inflammatory effects on leukocytes, such as triggering of chemotaxis, degranulation, synthesis of lipid mediators, and integrin activation, together with their critical role in the initiation and maintenance inflammatory diseases, and the recent identification of certain chemokine receptors as co-receptors for HIV-1 entry, have made chemokines and chemokine receptors an attractive new set of therapeutic targets.
Members of the chemokine family are produced and secreted by many cell types in response to early inflammatory mediators such as IL-1 xcex2 or TNFxcex1. The chemokine superfamily comprises two main branches: the xcex1-chemokines (or CXC chemokines) which are characterized by a single amino acid separating the first 2 cysteines, and the xcex2-chemokines (CC chemokines), which contain two adjacent cysteines. The xcex1-chemokine branch includes proteins such as IL-8, neutrophil activating peptide-2 (NAP-2), melanoma growth stimulatory activity (MGSA/gro or GROxcex1), and ENA-78, each of which have attracting and activating effects predominantly on neutrophils. The members of the xcex2-chemokine branch affect other cell types such as monocytes, lymphocytes, basophils, and eosinophils (Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648 (1991); Baggiolini, M., et al., Adv. Imunol., 55:97-179 (1994); Miller and Krangel, Crit. Rev. Immunol., 12:17-46 (1992); Jose, P. J., et al., J. Exp. Med., 179:881-118 (1994); Ponath, P. D., et al., J. Clin. Invest., 97:604-612 (1996)), and include proteins such as monocyte chemotactic proteins 1-4 (MCP-1, MCP-2, MCP-3, and MCP-4), RANTES, macrophage inflammatory proteins (MIP-1xcex1, MIP-1xcex2), thymus and activation-regulated chemokine (TARC) and macrophage-derived chemokine (MDC).
Chemokines bind to 7 transmembrane spanning G protein-coupled receptors (Murphy, P. M., Annu. Rev. Immunol, 12:593-633 (1994)). A number of xcex2 chemokine receptors (CCR1-CCR10) have been identified to date, and the search for additional chemokine receptors is the subject of active research (Baggiolini, Nature 392:565-568 (1998)). Chemokine receptor CCR1 was identified by Nomura et al., (Int. Immunol. 5:1239-1249 (1993); Neote et al., (Cell 72:415-425 (1993)) and Gao et al., (J. Exp. Med. 177:1421-1427 (1993)). CCR1 was originally found to signal in response to MIP-1xcex1 and RANTES, but more recently has also been shown to signal in response to additional chemokine ligands.
The selective recruitment of leukocyte subsets to sites of inflammation and the ordered trafficking of leukocytes through the circulation, tissues, lymphatic system and secondary lymphoid organs is controlled in part by the differential expression of chemokine receptors on subsets of cells. Such expression patterns would seem to ensure that a functionally related group of leukocytes can coordinately respond to a specific set of chemokines induced by a given stimulus. However, most leukocytes express several chemokine receptors, many with complex and promiscuous ligand interactions. For T cells, PCR or Northern blotting indicates that the known receptors for CC chemokines are expressed on subsets of T cells. Delineating exactly which subsets express particular receptors is an area of intense study, because chemokine receptor expression may explain the localization or migration of various cell types, such as TH1 or TH2 T cells or tissue homing subsets. It may also determine which T cells are infected with different strains of HIV-1. This makes elucidating the normal immune function for a specific receptor on a given cell type and determining the relevance to initiation and progression of disease difficult, especially since specific antibodies are not available for many chemokine receptors.
CCR1 was the first chemokine receptor shown to interact with Cxe2x80x94C (xcex2) chemokines. Originally identified as a MIP-1xcex1/RANTES receptor, more recent studies have demonstrated that CCR1 may have as many as 6 or more xcex2 chemokine ligands. Work described herein characterizes the expression of CCR1 by flow cytometry and assesses in vitro the relative functional contribution of this receptor on human leukocytes using a blocking monoclonal antibody. In peripheral blood, all monocytes express high levels of CCR1, and monocyte responses to both MIP-1xcex1 and RANTES can be completely blocked by the anti-CCR1 mAb 2D4. CCR1 is expressed on a small percentage of CD45RO+CD26+T cells, suggesting that it marks a subset of memory T cells. In contrast with chemokine receptors CCR5 and CXCR3, which are upregulated on activated T cells, CCR1 expression is diminished upon T cell activation. Neutrophils express low levels of CCR1 and show a weak response to MIP-1xcex1 in [Ca2+]i mobilization assays, which can be inhibited by the anti-CCR1 mAb 2D4. The expression of CCR1 on eosinophils is extremely variable among individuals, ranging from  greater than 90% positive to completely negative. The expression pattern of CCR1 suggests that it is involved in a broad range of immunological activities, playing a major role in monocyte and eosinophil function, as well as in the function of a subset of T lymphocytes.
The present invention relates to an antibody (immunoglobulin) or functional fragment thereof (e.g., an antigen-binding fragment) which binds to a mammalian CC-chemokine receptor 1 (also referred to as CCR1 or CKR-1) or portion of the receptor (anti-CCR1). In one embodiment, the antibody of the present invention or fragment thereof has specificity for human CCR1 or a portion thereof. In another embodiment, the antibody or fragment of the invention inhibits (reduces or prevents) binding of a ligand (e.g., MIP-1xcex1, RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1 or MPIF) to the receptor and inhibits one or more functions associated with binding of the ligand to the receptor (e.g., leukocyte trafficking). In a preferred embodiment, the ligand is MIP-1xcex1or RANTES. For example, as described herein, antibodies and fragments thereof of the present invention which bind human CCR1 or a portion thereof, can block binding of a chemokine (e.g., MIP-1xcex1, RANTES, MCP-2, MCP-3, leukotactin-1, HCC-1, or MPIF) to the receptor and inhibit function associated with binding of the chemokine to the receptor.
In a preferred embodiment, the antibody of the invention or fragment thereof has the same or similar epitopic specificity as monoclonal antibody (mAb) LS125-2D4 (2D4). For example, the antibody or fragment thereof can have epitopic specificity for the second extracellular loop of CCR1, such as a portion of CCR1 from about amino acid 171 to about amino acid 205. In one embodiment, the antibody is monoclonal antibody 2D4 or an antibody which can compete with 2D4 for binding to human CCR1 or a portion of human CCR1. Functional fragments of the foregoing antibodies are also envisioned.
In another embodiment, the antibody or fragment thereof binds CCR1 with an affinity of greater than about 5xc3x9710xe2x88x928 M, and more preferably at least about 5xc3x9710xe2x88x929 M. In a further embodiment, the antibody of the invention or fragment thereof inhibits chemokine binding to CCR1, preferably with an IC50 of less than about 10 xcexcg/ml, more preferably less than about 5 xcexcg/ml, and more preferably less than about 1.0 xcexcg/ml. In one embodiment, the antibody of the invention or fragment thereof inhibits MIP-1xcex1binding to CCR1 with an IC50 of about 0.5 xcexcg/ml. In another embodiment, the antibody of the invention or fragment thereof inhibits RANTES binding to CCR1 with an IC50 of about 0.7 xcexcg/ml. In a further embodiment of the invention, the antibody or fragment thereof inhibits of chemokine-induced (e.g., MIP-1xcex1-, RANTES- or HCC-1-induced) chemotaxis of cells (e.g., CCR1-bearing cells), preferably at less than about 50 xcexcg/ml, more preferably at less than about 20 xcexcg/ml, and even more preferably at less than about 10 xcexcg/ml. It is envisioned that any of the antibodies or fragments described herein can be used in the methods described herein.
The present invention also relates to an antibody or functional fragment thereof (e.g., an antigen-binding fragment) which binds to a mammalian CCR1 or portion of the receptor and provides increased fluorescent staining intensity of CCR1 or compositions comprising CCR1 relative to other anti-CCR1 antibodies. In one embodiment, the antibody is monoclonal antibody 2D4 or an antibody which can compete with 2D4 for binding to human CCR1 or a portion of human CCR1.
The present invention further relates to a method of inhibiting the interaction of a cell bearing mammalian (e.g., human, non-human primate or murine) CCR1 with a ligand thereof, comprising contacting the cell with an effective amount of an antibody or functional fragment thereof which binds to a mammalian CCR1 or a portion of CCR1. Suitable cells include granulocytes, leukocytes, such as monocytes, macrophages, basophils and eosinophils, mast cells, and lymphocytes including T cells (e.g., CD8+ cells, CD4+ cells, CD26+ cells, CD25+ cells, CD45RO+ cells) such as Th1 and Th2 cells, and other cells expressing CCR1, such as a recombinant cell expressing CCR1 or portion thereof (e.g., transfected cells). In a particular embodiment, the antibody is 2D4 or an antibody which can compete with 2D4 for binding to human CCR1 or a portion of human CCR1.
Another embodiment of the invention relates to a method of inhibiting the interaction of a cell bearing mammalian CCR1 with a chemokine, comprising contacting said cell with an effective amount of an antibody or functional fragment thereof which binds to CCR1 or a portion of said receptor. In one embodiment of the method, the antibody or functional fragment thereof is any one or more of 2D4, an antigen-binding fragment of 2D4 or an antibody or fragment thereof having an epitopic specificity which is the same as or similar to that of 2D4. Furthermore, the invention relates to a method of inhibiting a function associated with binding of a chemokine to CCR1, comprising administering an effective amount of an antibody or functional fragment thereof which binds to mammalian CCR1 or a portion of said receptor. In one aspect of the method, the antibody or functional fragment thereof is any one or more of 2D4, an antigen-binding fragment of 2D4, or an antibody or fragment thereof having an epitopic specificity which is the same as or similar to that of 2D4.
Another aspect of the invention is a method of identifying expression of a mammalian CCR1 or portion of the receptor by a cell. According to the method, a composition comprising a cell or fraction thereof (e.g., a membrane fraction) is contacted with an antibody or functional fragment thereof (e.g., 2D4) which binds to a mammalian CCR1 protein or portion of the receptor under conditions appropriate for binding of the antibody thereto, and the formation of a complex between said antibody or fragment and said protein or portion thereof is detected. Detection of the complex, directly or indirectly, indicates the presence of the receptor or portion thereof on the cell or fraction thereof. The present invention also relates to a kit for use in detecting the presence of CCR1 or a portion thereof in a biological sample, comprising an antibody or functional fragment thereof which binds to a mammalian CCR1 or a portion of said receptor, and one or more ancillary reagents suitable for detecting the presence of a complex between said antibody or fragment and said protein or portion thereof.
Also encompassed by the present invention are methods of identifying additional ligands or other substances which bind a mammalian CCR1 protein, including inhibitors and/or promoters of mammalian CCR1 function. For example, agents having the same or a similar binding specificity as that of an antibody of the present invention or functional fragment thereof can be identified by a competition assay with said antibody or fragment. Thus, the present invention also encompasses methods of identifying ligands or other substances which bind the CCR1 receptor, including inhibitors (e.g., antagonists) or promoters (e.g., agonists) of receptor function. In one embodiment, cells which naturally express CCR1 receptor protein or suitable host cells which have been engineered to express a CCR1 receptor or variant encoded by a nucleic acid introduced into said cells are used in an assay to identify and assess the efficacy of ligands, inhibitors or promoters of receptor function. Such cells are also useful in assessing the function of the expressed receptor protein or polypeptide.
Thus, the invention also relates to a method of detecting or identifying an agent which binds a mammalian CCR1 or ligand binding variant thereof, comprising combining an agent to be tested, an antibody or antigen-binding fragment of the present invention (e.g., monoclonal antibody 2D4, an antibody having an epitopic specificity which is the same as or similar to that of 2D4, antigen-binding fragments of 2D4) and a composition comprising a mammalian CCR1 protein or a ligand binding variant thereof. The foregoing components can be combined under conditions suitable for binding of the antibody or antigen-binding fragment to mammalian CCR1 protein or a ligand binding variant thereof, and binding of the antibody or fragment to the mammalian CCR1 protein or ligand binding variant is detected or measured, either directly or indirectly, according to methods described herein or other suitable methods. A decrease in the amount of complex formed relative to a suitable control (e.g., in the absence of the agent to be tested) is indicative that the agent binds said receptor or variant. The composition comprising a mammalian CCR1 protein or a ligand binding variant thereof can be a membrane fraction of a cell bearing recombinant CCR1 protein or ligand binding variant thereof. The antibody or fragment thereof can be labeled with a label such as a radioisotope, spin label, antigen label, enzyme label, fluorescent group and chemiluminescent group. These and similar assays can be used to detect agents, including ligands (e.g., chemokines which interact with CCR1) or other substances, including inhibitors or promoters of receptor function, which can bind CCR1 and compete with the antibodies described herein for binding to the receptor.
According to the present invention, ligands, inhibitors or promoters of receptor function can be identified in a suitable assay, and further assessed for therapeutic effect. Inhibitors of receptor function can be used to inhibit (reduce or prevent) receptor activity, and ligands and/or promoters can be used to induce (trigger or enhance) normal receptor function where indicated. These ligands, inhibitors and promoters can be used to treat inflammatory diseases, autoimmune diseases, atherosclerosis, and graft rejection, or HIV infection, for example, in a method comprising administering an inhibitor of receptor function (e.g., chemokine binding or HIV binding) to an individual (e.g., a mammal, such as a human). These ligands, inhibitors and promoters can also be used in a method of stimulating receptor function by administering a novel ligand or promoter to an individual, providing a new approach to selective stimulation of leukocyte function, which is useful, for example, in the treatment of infectious diseases and cancer.
The present invention also encompasses a method of inhibiting leukocyte trafficking in a patient, comprising administering to the patient an effective amount of an antibody or functional fragment thereof which binds to a mammalian CCR1 or portion of said receptor and inhibits function associated with binding of a ligand to the receptor.
The present invention also relates to a method of inhibiting or treating CCR1-mediated disorders, such as inflammatory disorders, comprising administering to a patient an effective amount of an antibody or functional fragment thereof which binds to a mammalian CCR1 or portion of said receptor and inhibits CCR1-mediated function.
The present invention further relates to an antibody or fragment thereof as described herein (e.g., monoclonal antibody 2D4, an antigen-binding fragment of 2D4, an antibody having an epitopic specificity which is the same as or similar to that of 2D4) for use in therapy (including prophylaxis) or diagnosis, and to the use of such an antibody or fragment for the manufacture of a medicament for the treatment of a CCR1-mediated disorder, or other disease or inflammatory condition as described herein.